Saponin-decomposing enzyme, gene thereof and large-scale production system for producing soyasapogenol B

ABSTRACT

The present invention provides a protein having saponin-decomposing activity, more specifically a protein which can decompose a glycoside having soyasapogenol B as an aglycone to produce soyasapogenol B, a polynucleotide encoding such a protein, and a method of producing soyasapogenol B on a large scale using the same. A protein according to the present invention are concerned with (a), (b) or (c), namely (a) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 2, 4, and 6; (b) a protein that has at least 50% homology to the protein comprising the amino acid sequence of the sequence described in (a) and having saponin-decomposing activity; or (c) a protein comprising a modified amino acid sequence of the sequence described in (a) that has one or more amino acid residues deleted, substituted, inserted, or added and having saponin-decomposing activity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel saponin-decomposing enzyme, agene thereof, and a novel method for producing soyasapogenol B usingthem.

2. Background Art

Soyasapogenol B (12-oleanane-3,22,24-triol) is one of the aglycones ofsaponins contained in legumes and has been reported to have variousphysiological activities since early times. For example, plateletaggregation suppressing effect, anticomplementary activity, andpreventive and therapeutic activity for nephritis, rheumatism, immunediseases such as systemic lupus erythematosus, autoimmune diseases orthrombosis have been reported (Chem. Pharm. Bull., 24, 121-129, 1976;Chem. Pharm. Bull., 30, 2294-2297, 1982; Kagaku to Seibutsu, 21,224-232, 1983; Japanese Patent Application Laid-open No. 37749/1986).Further, growth-suppressing effect on cells derived from human coloncancer and human ovarian cancer has been reported (Japanese PatentApplication Laid-open No. 37749/1986; Japanese Patent ApplicationLaid-open No. 234396/1998).

Soyasapogenol B can be produced, for example, by chemically hydrolyzingsugar chains of saponins contained in soybean seeds as glycosides(soyasaponins I-V). However, this is not an effective production methodbecause a considerable number of by-products may be produced dependingon the conditions for acid hydrolysis. Further, soybean seeds are knownalso to contain saponins which have soyasapogenol A (soyasaponins A1-A6)or soyasapogenol E as an aglycone. Therefore, when soyasapogenol B isprepared from soybeans, the resulting preparation may easily containsoyasapogenol A and soyasapogenol E as impurities so that it isdifficult to purify soyasapogenol B alone from such preparation.Further, since the saponin content of soybean seeds is generally as lowas about 0.2% (Yakugaku Zasshi, 104, 162-168, 1984), there is a need formore efficient production.

As for methods of producing soyasapogenol B using microorganisms, amethod with genus Streptomyces (Chem. Pharm. Bull. 32: 1287-1293, 1984)and a method with genus Penicillium (Japanese Patent ApplicationLaid-open No. 234396/1998) is known. However, these methods of producingsoyasapogenol B using microorganisms are poor in productivity andpracticality.

Further, it has been reported that soyasapogenol B can be obtained as aby-product in a method in which an acid oligosaccharide havingglucuronic acid as the reduced end is produced by hydrolyzing aglucuronide saponin using the enzyme (glucuronidase) produced bymicroorganisms that belong to genus Aspergillus or a culture containingthis enzyme (Japanese Patent Publication No. 32714/1995). However, thismethod is primarily a method of producing acid oligosuccharides, andonly a qualitative confirmation of soyasapogenol B is described in thisreport. Further, this report revealed the molecular weight of the enzymehaving activity of interest but not the amino acid sequence thereof.

On the other hand, the search for microorganisms which efficientlyproduce soyasapogenol B by selectively hydrolyzing a glycoside havingsoyasapogenol B as an aglycone resulted in finding filamentous fungusstrains that belong to genus Neocosmospora or genus Eupenicillium. Ithas been found that soyasapogenol B is produced and accumulated in aculture medium at a high concentration by culturing filamentous fungi,that belong to genus Neocosmospora or genus Eupenicillium, in a mediumcontaining a saponin (a glycoside having soyasapogenol B as an aglycone)(see WO 01/81612).

Examples of such filamentous fungi include Neocosmospora vasinfecta var.vasinfecta PF1225 that belongs to genus Neocosmospora and Eupenicilliumbrefeldianum PF1226 that belongs to genus Eupenicillium (see WO01/81612).

Soyasapogenol B of interest can be produced using such fungi as theyare, depending on the amount of saponins added to the medium. However,the amount of saponins to be added to the medium is limited because ofthe surface-active property of saponins, which easily foam. Further,viscosity of the medium supplemented with saponins is expected toincrease because of the surface-active property. Accordingly, in orderto improve the yield in producing the target substance from the culture,the extraction process has to be repeated several times. Further,soybean extract, which is generally used as a natural resource toeffectively supply saponins, usually contains components other thansaponins, such as lipids, proteins and polysaccharides. Therefore, thepossible amount of saponins to be added to a medium ultimately dependson the purity of the soybean extract, which does not necessarily assureefficient production.

There is a need to develop a method for the large scale production ofsoyasapogenol B by an enzyme reaction using a saponin-decomposing enzymeproducing enzyme, in which soyasapogenol B is efficiently produced and ahigh yield is maintained independently of the saponin content in asoybean extract, contrary to conventional methods.

SUMMARY OF THE INVENTION

Recently, the present inventors succeeded in isolating and purifying aprotein having saponin-decomposing activity from microorganisms havingsaponin-decomposing activity (occasionally called “saponin-decomposingenzyme” hereinafter) and in identifying a gene encoding this protein.Further, the present inventors were able to obtain a highly activesaponin-decomposing enzyme by expressing the resulting gene in aheterologous host. Further, the present inventors were able toeffectively produce soyasapogenol B by carrying out an enzyme reactionusing the saponin-decomposing enzyme thus obtained. The presentinvention is based on these findings.

Accordingly, an objective of the present invention is to provide aprotein having saponin-decomposing activity, more specifically a proteinwhich can decompose a glycoside having soyasapogenol B as an aglycone toproduce soyasapogenol B, a polynucleotide encoding such protein, and amethod of producing soyasapogenol B on a large scale using the same.

A protein according to the present invention is selected from the groupconsisting of the followings:

-   -   (a) a protein comprising an amino acid sequence selected from        the group consisting of the amino acid sequences shown in SEQ ID        NOs: 2, 4, and 6;    -   (b) a protein that has at least 50% homology to the protein        comprising the amino acid sequence of the sequence described        in (a) and having saponin-decomposing activity; and    -   (c) a protein comprising a modified amino acid sequence of the        sequence described in (a) that has one or more amino acid        residues deleted, substituted, inserted, or added and having        saponin-decomposing activity.

A polynucleotide according to the present invention is selected from thegroup consisting of the followings:

-   -   (i) a polynucleotide consisting of a DNA sequence selected from        the group consisting of the DNA sequences of SEQ ID NOs: 1, 3,        and 5;    -   (ii) a polynucleotide that has at least 70% homology to the        polynucleotide consisting of the DNA sequence of (i) and encodes        a protein having saponin-decomposing activity;    -   (iii) a polynucleotide consisting of a modified DNA sequence of        the sequence described in (i) that has one or more bases        deleted, substituted, inserted, or added and encodes a protein        having saponin-decomposing activity; and    -   (iv) a polynucleotide that hybridizes with a polynucleotide        comprising the DNA sequence described in (i) under stringent        conditions and encodes a protein having saponin-decomposing        activity.

A recombinant vector according to the present invention comprises apolynucleotide of the present invention.

Further, a host according to the present invention is a host transformedwith the abovementioned recombinant vector.

A process for producing a protein of interest according to the presentinvention comprises culturing the abovementioned transformed host andcollecting a protein having saponin-decomposing activity from theresulting culture.

According to the present invention, a highly active saponin-decomposingenzyme can be obtained. Further, by using this enzyme, soyasapogenol Bcan be obtained efficiently and on a large scale from saponin. Accordingto this method, soyasapogenol B can be obtained independently of thesaponin content of, for example, a soybean extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction and restriction map for plasmid pCB-SBe.

FIG. 2 shows the optimum pH for recombinant saponin-decomposing enzymesin Example 5. In the Figure, the wild-type SDN means thesaponin-decomposing enzyme derived from Neocosmospora vasinfecta var.vasinfecta PF1225, and the recombinant SDN means the recombinantsaponin-decomposing enzyme.

FIG. 3 shows the optimum temperature for recombinant saponin-decomposingenzymes in Example 5. In the Figure, the wild-type SDN means thesaponin-decomposing enzyme derived from Neocosmospora vasinfecta var.vasinfecta PF1225, and the recombinant SDN means the recombinantsaponin-decomposing enzyme.

FIG. 4 shows the construction and restriction map for plasmid pCB-SDAe.

FIG. 5 shows the optimum pH for recombinant saponin-decomposing enzymesin Example 8. In the Figure, the wild-type SDA means thesaponin-decomposing enzyme derived from Aspergillus sp. PF1224, and therecombinant SDA means the recombinant saponin-decomposing enzyme.

FIG. 6 shows the optimum temperature for recombinant saponin-decomposingenzymes in Example 8. In the Figure, the wild-type SDA means thesaponin-decomposing enzyme derived from Aspergillus sp. PF1224, and therecombinant SDA means the recombinant saponin-decomposing enzyme.

FIG. 7 shows the construction and restriction map for plasmid pCB-SDEs.

FIG. 8 shows the optimum pH for recombinant saponin-decomposing enzymesin Example 12. In the Figure, the wild-type SDE means thesaponin-decomposing enzyme derived from Eupenicillium brefeldianumPF1226, and the recombinant SDN means the recombinantsaponin-decomposing enzyme.

FIG. 9 shows the optimum temperature for recombinant saponin-decomposingenzymes in Example 12. In the Figure, the wild-type SDE means thesaponin-decomposing enzyme derived from Eupenicillium brefeldianumPF1226, and the recombinant SDE means the recombinantsaponin-decomposing enzyme.

FIG. 10 shows the result made a comparison with SDN, SDA and SDE bysearching their homology each other.

DETAILED DESCRIPTION OF THE INVENTION Deposition of Microorganisms

The strain Neocosmospora vasinfecta var. vasinfecta PF1225 was depositedwith the International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-5466, dated Mar. 13, 2000(original deposition date). The accession number is FERM BP-7475.

The strain Eupenicillium brefeldianum PF1226 was deposited with theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, AIST Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan 305-5466, dated Mar. 13, 2000 (originaldeposition date). The accession number is FERM BP-7476.

The strain Aspergillus sp. PF1224 was deposited with the InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan 305-5466, dated May 24, 2001. The accession number isFERM BP-8004.

The strain Trichoderma viride MC300-1 was deposited with theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, AIST Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan 305-5466, dated Sep. 9, 1996 (originaldeposition date). The accession number is FERM BP-6047.

Protein Having Saponin-Decomposing Activity

A saponin-decomposing enzyme isolated and purified from Neocosmosporavasinfecta var. vasinfecta PF1225 (FERM BP-7475) was revealed to be anovel enzyme system since it has no homology to any saponin-decomposingenzyme found to date and is different from glucronidase derived from amicroorganism belonging to genus Aspergillus (a glycoprotein having amolecular weight of about 158,000 consisting of subunits each having amolecular weight of 35,000 and 45,000 described in Japanese PatentPublication No. 32714/1995), which decomposes glucronide saponin, in itssubunit structure and molecular weight.

Further, the protein according to the present invention and glucronidasepreviously disclosed in the Patent Publication were studied for theiridentity and homology. Since the strain belonging to genus Aspergillusdescribed in said Patent Publication was not readily available, anotherstrain belonging to genus Aspergillus was used. The strain used wasAspergillus sp. PF1224, which was identified to be a filamentous fungus,Deuteromycetes, belonging to genus Aspergillus according to themicrobial properties shown below.

(1) Colony Features

Colonies grow well on a Czapek's yeast extract agar medium at 25° C.attaining a diameter of 80 mm in 7 days. The colonies are yellow toyellowish green, woolly and rich in conidia and sclerotia. The reverseside becomes pale brown. Colonies grow well on a malt extract agarmedium at 25° C. attaining a diameter of 80 mm in 7 days.

The colonies are yellow to yellowish green, woolly and rich in conidiaand sclerotia. The reverse side becomes ocherous. Colonies are slightlysuppressed on either medium when cultured at 37° C.

(2) Morphological Features

Conidial heads are yellow to yellowish green and radiate to loosecylindrical. Conidiophores are rough and colorless and vesicles arerodlike to subglobose, bearing aspergilla on almost the entire surface.Monoseriate and biseriate aspergilla are mixed; they are generallybiseriate. Metulae are 8-12×4-5 μm and phialides are 8-12×3-4 μm.Conidia are globose to subglobose, rough, and 4-6 μm in length.

The enzyme referred to as glucronidase was confirmed using Aspergillussp. PF1224, which revealed that the saponin-decomposing enzyme isolatedand purified from Aspergillus sp. PF1224 by the present inventors has amolecular weight of 90 kDa, an optimum pH of 5 to 6, and an optimumtemperature of 45° C. to 50° C. (see Reference Example). Furthermore, itwas revealed that the saponin-decomposing enzyme isolated and purifiedfrom Eupenicillium brefeldianum PF1226 has a molecular weight of 90 kDa,an optimum pH of 5 to 6, and an optimum temperature of 40° C. to 45° C.

The protein of the present invention and the glucuronidase described inthe abovementioned Patent Publication were compared for their molecularweight and subunit configuration since information such as an amino acidsequence of glucronidase was not disclosed in this Patent Publication tocompare homology of the amino acid sequence or the like. Results showedthat the protein according to present invention is a protein differentfrom the glucronidase described in the Patent Publication.

As mentioned above, the present invention provides a protein selectedfrom the group consisting of:

-   -   (a) a protein comprising an amino acid sequence selected from        the group consisting of the amino acid sequences shown in SEQ ID        NOs: 2, 4, and 6;    -   (b) a protein that has at least 50% homology to the protein        comprising the amino acid sequence of the sequence described        in (a) and having saponin-decomposing activity; and    -   (c) a protein comprising a modified amino acid sequence of the        sequence described in (a) that has one or more amino acid        residues deleted, substituted, inserted, or added and having        saponin-decomposing activity.

Namely, a protein according to the present invention comprises an aminoacid sequence which is identical or substantially identical to the aminoacid sequence shown in SEQ ID NO: 2, 4, or 6.

An amino acid sequence that is substantially identical to the amino acidsequence shown in SEQ ID NO: 2, 4, or 6 herein means an amino acid thatis typically more than 50%, preferably more than 70%, more preferablymore than 80%, further preferably more than 90%, furthermore preferablymore than 95%, or most preferably more than 98% homologous to any one ofthe amino acid sequences shown in these SEQ ID NOs.

Further, these figures for homology shown in the present specificationcan be any figures calculated using a homology search program known tothe skilled in the art. For example, figures can be readily calculatedusing default parameters in FASTA, BLAST, or the like.

For example, when the figures for homology has been calculated usingdefault parameters in the homology search program Genetyx (manufacturedby Genetyx Co.), the figure for homology between SDN and SDA is 51%,that between SDA and SDE is 52%, and that between SDE and SDN is 51%.

Further, a protein comprising an amino acid sequence that issubstantially identical to the amino acid sequence shown in SEQ ID NO:2, 4, or 6 means a protein comprising a modified amino acid sequencethat has one or more amino acid residues deleted, substituted, inserted,or added and having saponin-decomposing activity.

The number of amino acid residues that can be deleted, substituted,inserted, or added is preferably 1 to 50, more preferably 1 to 30,further preferably 1 to 10, furthermore preferably 1 to 5, and mostpreferably 1 to 2.

In a more preferred embodiment of the invention, the protein describedin (b) above is a protein comprising a modified amino acid sequence thathas one or more conservatively substituted amino acid residues in theamino acid sequence (a) above and having saponin-decomposing activity.

The expression “conservatively substituted” herein means that one ormore amino acid residues are substituted with other amino acid residueswhich are chemically homologous not to substantially alter the proteinactivity. For example, a hydrophobic residue is substituted with anotherhydrophobic residue or a polar residue is substituted with another polarresidue having the same electric charge. Functionally homologous aminoacids of different types, which can be conservatively substituted inthis way, are known to the skilled in the art. Examples of such aminoacids include non-polar (hydrophobic) amino acids such as alanine,valine, isoleucine, leucine, proline, tryptophan, phenyalanine, andmethionine; polar (neutral) amino acids such as glycine, serine,threonine, tyrosine, glutamine, asparagine, and cysteine; positivelycharged (basic) amino acids such as arginine, histidine, and lysine; andfurther, negatively charged (acidic) amino acids such as aspartic acid,and glutamic acid.

In the present invention, the term “protein having saponin-decomposingactivity” means a protein that is verified to have an activity todecompose saponin. For example it means a protein which is verified tohave saponin-decomposing activity when measured under the sameconditions as described in Example 5. This protein can be isolated andpurified from “organisms having saponin-decomposing activity” describedbelow.

A protein according to the present invention can be obtained, forexample, as follows.

An organisms having saponin-decomposing activity is cultured and aprotein having saponin-decomposing activity is isolated and purifiedfrom the resulting culture using the saponin-decomposing activity as anindex. The amino acid sequence of the protein thus purified is analyzed,an oligonucleotide encoding this sequence is synthesized, and the PCR(polymerase chain reaction) is carried out using DNA of said organism asa template to synthesize a long probe. A DNA sequence of the translationregion of the saponin-decomposing enzyme gene is analyzed by the inversePCR or the RACE (rapid amplification of cDNA ends) method using thisprobe. The translation region of the saponin-decomposing enzyme thusobtained is linked to a regulatory sequence which functions in a host tobe used for expression to obtain an expression vector. This expressionvector is used to transform the host, the resulting transformant iscultured, and thus a saponin-decomposing enzyme can be obtained.

An “organism having saponin-decomposing activity” herein can be anyorganism having saponin-decomposing activity and is not particularlylimited and includes microorganisms and plants. Examples of suchmicroorganism include filamentous fungi that belong to genusNeocosmospora, genus Aspergillus, and genus Eupenicillium. These fungiare used in manufacturing soy sauce and soybean paste by fermentationand known to have saponin-decomposing activity. Further, saidmicroorganisms include actinomycetes and bacteria since some of them mayalso have saponin-decomposing activity. Said plants include plantitself, plant cells, callus or culture cells derived from leguminousplants since some saponin glycotransferase of such plants may catalyzereverse reaction.

In a preferred embodiment of the present invention, a protein or apolynucleotide according to the present invention is derived from amicroorganism, more preferably a microorganism belonging to filamentousfungus. A microorganism belonging to such filamentous fungus ispreferably filamentous fungus belonging to genus Neocosmospora, genusAspergillus, or genus Eupenicillium.

Examples of filamentous fungi that belong to genus Neocosmospora includeNeocosmospora vasinfecta var. vasinfecta PF1225 (accession number: FERMBP-7475) and mutants thereof. Examples of filamentous fungi that belongto genus Aspergillus include Aspergillus sp. PF1224 (accession number:FERM BP-8004) and mutants thereof. Examples of filamentous fungi thatbelong to genus Eupenicillium include Eupenicillium brefeldianum PF1226(accession number: FERM BP-7476) and mutants thereof.

Polynucleotide

The present invention provides a polynucleotide which encodes a proteinof the present invention.

A polynucleotide according to the present invention is typically apolynucleotide selected from the group consisting of (i) to (iv)described above.

Namely, according to one embodiment of the present invention, thepolynucleotide comprises a DNA sequence selected from the groupconsisting of the DNA sequences shown in SEQ ID NOs: 1, 3, and 5.

According to another embodiment of the present invention, thepolynucleotide comprises a DNA sequence having at least 70% homology tothe polynucleotide comprising the DNA sequence shown in SEQ ID NO: 1, 3,or 5 and encodes a protein having saponin-decomposing activity. Thehomology to the polynucleotide comprising the DNA sequence shown in SEQID NO: 1, 3, or 5 is preferably more than 80%, more preferably more than90%, furthermore preferably more than 95%, or most preferably more than98%.

Further, figures for homology shown in the present specification can beany figures calculated using a homology search program known to theskilled in the art. For example, figures can be readily calculated usingdefault parameters in FASTA, BLAST, or the like.

According to another embodiment of the present invention, thepolynucleotide comprises a DNA sequence having one or more basesdeleted, substituted, inserted, or added in the DNA sequence shown inSEQ ID NO: 1, 3, or 5 and encodes a protein having saponin-decomposingactivity.

Here, the number of amino acid residues that can be deleted,substituted, inserted, or added is preferably 1 to 50, more preferably 1to 30, further preferably 1 to 10, furthermore preferably 1 to 5, andmost preferably 1 to 2.

According to still another embodiment of the present invention, thepolynucleotide hybridizes with a polynucleotide comprising the DNAsequence shown in SEQ ID NO: 1, 3, or 5 under stringent conditions andencodes a protein having saponin-decomposing activity. Further,according to the present invention, the polynucleotide also implies apolynucleotide which is complementary to a polynucleotide encoding aprotein having saponin-decomposing activity.

The term “stringent conditions” herein means controlled conditions underwhich a probe comprising a DNA sequence partly or entirely encoding anamino acid sequence of a protein according to the present inventionhybridized with a gene encoding a corresponding homologue while thisprobe does not hybridize with glucronidase having a molecular weightdescribed in Japanese Patent Publication No. 32714/1995. Morespecifically, for example, according to the method of ECL Direct DNA/RNALabeling Detection System (Amersham) using the whole length ofpolynucleotide encoding the standardized amino acid sequence shown inSEQ ID NO: 1, 3, or 5 as a probe, pre-hybridization is first carried outfor 1 hour (42° C.), after which said probe is added, hybridization (42°C.) is carried out for 15 hours, and then washing process is carried outfirst with a 0.5×SSC solution (SSC: 15 mM trisodium citrate, 150 mMsodium chloride) supplemented with 0.4% SDS and 6 M urea twice at 42° C.for 20 minutes and then with a 5×SSC solution twice at room temperature(about 25° C.) for 10 minutes.

Recombinant Vector

The present invention provides a recombinant vector comprising theabovementioned polynucleotide.

The procedure and method for constructing a recombinant vector accordingto the present invention can be any of those commonly used in the fieldof genetic engineering.

Examples of the expression vector as used herein include vectors whichcan be incorporated into a host chromosome DNA and vectors having aself-replicable autonomous replication sequence which can be present asa plasmid in a host cell, for example, pUC vectors (e.g., pUC18 andpUC118), pBluescript vectors (e.g., pBluescript II KS+), and plasmidssuch as pBR322 plasmid. One or more of copies of the gene can be presentin a host cell.

A regulatory sequence for the recombinant vector can be any regulatorysequence which can function in a host and is not particularly limited.For example, a promoter, a terminator, and the like can be used. Such aregulatory sequence can be ligated to a gene encoding a protein havingsaponin-decomposing activity for the gene expression.

The ligation to a regulatory sequence can be carried out, for example,according to an ordinary method by inserting a translation region of agene encoding a protein of interest (gene of interest) downstream of apromoter in the right direction. In this case, the protein can beexpressed as a fusion protein by ligating the gene of interest to aforeign gene encoding a translation region of another protein.

The expressed protein having saponin-decomposing activity or theexpressed fused protein having said activity can be produced in a hostcell used for expression or released into a medium.

For example, a saponin-decomposing enzyme derived from Neocosmosporavasinfecta var. vasinfecta PF1225 (FERM BP-7475) was revealed to have asignal peptide sequence of 26 amino acid residues at the N-terminal sideaccording to the DNA sequence analysis and N-terminal amino acidsequence analysis (see Example). Similarly, a saponin-decomposing enzymederived from Aspergillus sp. PF1224 and a saponin-decomposing enzymederived from Eupenicillium brefeldianum PF1226 were revealed to havesignal peptide sequences of 28 amino acid residues and 17 amino acidresidues at the N-terminal side, respectively.

Accordingly, for example, when filamentous fungi such as those belongingto genus Trichoderma and genus Aspergillus are used as a host, theprotein can be released into a medium by utilizing a signal sequenceincluded in this sequence.

Further, the saponin-decomposing enzyme derived from Neocosmosporavasinfecta var. vasinfecta PF1225 having a molecular weight of about 77kDa is inferred to be glycoproteins from a molecular weight of about 68kDa estimated from a deduced amino acid composition and a molecularweight of about 68 kDa of a protein expressed in strains of Escherichiacoli and Trichoderma viride. Similarly, the saponin-decomposing enzymederived from Aspergillus sp. PF1224 having a molecular weight of about90 kDa is inferred to be glycoproteins from a molecular weight of about65 kDa estimated from a deduced amino acid composition and a molecularweight of about 80 kDa of a protein expressed in strains of Trichodermaviride. Further, the saponin-decomposing enzyme derived fromEupenicillium brefeldianum PF1226 having a molecular weight of about 90kDa is inferred to be glycoproteins from a molecular weight of about 65kDa estimated from a deduced amino acid composition.

These sugar chain are presumed not to have great influence on theexpression of activity. However, various modification after translation,such as addition of various sugar chains, can be carried outanticipating effective changes in heat resistance, optimum pH, stabilityduring storage, or the like.

A recombinant vector according to the present invention can beconstructed by further ligating a selective marker gene such as a drugresistance gene and/or a gene complementing a nutritional requirement.

A gene marker can be appropriately selected depending on the techniquefor selecting a transformant. For example, a gene encoding drugresistance or a gene complementing a nutritional requirement can beused. Examples of the drug resistance gene include genes conferringresistance to destomycin, benomyl, oligomycin, hygromycin, G418,pleomycin, bialaphos, blastcidin S, phleomycin, phosphinothricin,ampicillin, streptomycin, and kanamycin. Examples of the genecomplementing a nutritional requirement include amdS, pyrG, argB, trpC,niaD, TRP1, LEU2, and URA3. Further, a gene marker can be a genecomplementing a nutrient requirement indigenous to a host to be used forexpression in systems for synthesizing various amino acids, vitamins,nucleic acids, or the like, or a gene complementing a nutrientrequirement that is rendered by various mutagenic treatments.

Production of Transformant and Protein of Interest

The present invention provides a host transformed with theabovementioned recombinant vector.

A host to be used in the present invention is not particularlyrestricted and any organism which can properly transcript and translatea gene encoding a protein having saponin-decomposing activity can beused. Examples of the host include bacteria such as Escherichia coli andBacillus spp., actinomycetes, yeasts, filamentous fungi such asTrichoderma spp. and mutants thereof.

A recombinant vector for the gene expression can be introduced into ahost by an ordinary method. Examples of the method for the introductioninclude the electroporation method, the polyethylene glycol method, theaglobacterium method, the lithium method, and the calcium chloridemethod. A method effective to each host cell can be selected.

A transformant (transformed host cell) can be cultured according to anordinary method by appropriately selecting a medium, culture conditionsand the like.

Conventional components can be used in a medium. As a carbon source,glucose, sucrose, cellulose, starch syrup, dextrin, starch, glycerol,molasses, animal and vegetable oils, and the like can be used. As anitrogen source, soybean powder, wheat germ, cornsteep liquor, cottonseed lees, bouillon, peptone, yeast extract, ammonium sulfate, potassiumnitrate, urea, and the like can be used. If necessary, sodium,potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid,sulfuric acid, and other inorganic salts that can produce ions, such aspotassium chloride, magnesium sulfate, monopotassium phosphate, zincsulfate, manganese sulfate, and copper sulfate, can be effectivelyadded. If necessary, various vitamins, amino acids, trace nutrients suchas nucleotides, and selective drugs such as antibiotics can be added.Further, organic and inorganic substances to promote the growth oftransformants and enhance the expression of an introduced gene can beappropriately added.

Cultivation can be carried out in a medium selectively containing thesecomponents.

For example, in a liquid medium, the cultivation can be carried outusing a culture method under an aerobic condition, a shaking culturemethod, an agitation culture method with aeration, a submerged culturemethod or the like. The pH of the medium is, for example, about 5 to 8.The cultivation can be carried out at a normal temperature, such as 14°C. to 40° C., preferably 26° C. to 37° C., for about 1 to 25 days.

In a method of producing a protein of interest according to the presentinvention, a gene expression product, namely the protein of interesthaving saponin-decomposing activity, can be obtained from the culture oftransformed cells. The protein of interest can be obtained from theculture according to an ordinary method. For example, steps of theextraction from the culture (e.g., by mashing, and crushing underpressure), the recovery (e.g., by filtration and centrifugation), and/orthe purification (e.g., by salting out and solvent precipitation) can beappropriately combined. Furthermore, in these steps, a proteaseinhibitor, such as phenylmethylsulfonyl fluoride (PMSF), benzamidine andleupeptin, can be added if necessary.

According to another embodiment of the present invention, it is alsopossible to express a gene encoding a protein having saponin-decomposingactivity in a plant which produces saponins, such as plants of soybean,kidney bean, cowpea, pea, peanut, and broad bean, and alfalfa togenerate a plant body containing soyasapogenol B, from whichsoyasapogenol B is directly obtained. In this case, actin, ubiquitin,cauliflower mosaic virus 35S promoter, or the like, or a regulatorysequence of a gene specifically expressed at a part, such as the seed,can be used.

A gene encoding a protein having saponin-decomposing activity isproperly linked to such a regulatory sequence and further, a drugresistance gene conferring resistance to bialaphos, kanamycin,blastcidin S, or the like is linked if necessary. The resultant productcan be introduced into a plant cell, for example, by a directintroduction method such as the particle-gun method, the PEG method, theelectroporation method, and the microinjection method, or by an indirectintroduction method using Ti plasmid vector of aglobacterium to generatea transformed plant cell. Introduction of the gene into a plant cell orplant body can be carried out according the method of Vaeck M. et al(Nature, 328, 33-37, 1987).

The plant cell thus transformed can be redifferentiated by the methodknown to the skilled in the art into a complete body of a transformedplant. Further, the transformed plant is cultivated and the resultantwhole plants and/or organs, such as seeds, in which a gene encoding aprotein having saponin-decomposing activity is expressed are harvested,from which soyasapogenol B can be obtained using a method suitable forits property, such as the solvent extraction method.

Production of Soyasapogenol B

Another embodiment of the present invention provides a method ofproducing soyasapogenol B which comprises decomposing a glycoside havingsoyasapogenol B as an aglycone using a culture containing a proteinhaving saponin-decomposing activity which can be obtained from theabovementioned transformed host.

Further, still another embodiment of the present invention provides amethod of producing soyasapogenol B which comprises decomposing aglycoside having soyasapogenol B as an aglycone using at least one kindof protein selected from the group consisting of the abovementionedprotein and the protein which can be obtained from the abovementionedtransformed host.

Examples of the “glycoside having soyasapogenol B as an aglycone”include soyasaponins I, II, III, IV, and V; azukisaponins II and V,astragaloside VIII, and sophoraflavoside I, which are primarily found inleguminous plants.

Examples of the substance containing a glycoside having soyasapogenol Bas an aglycone include a substance extracted from soybeans or defattedsoybeans (soybean cake) with hot water, alcohol or alcohol hydrate, orpreferably a substance from which impurities such as proteins, sugarsand lipids are removed by an ordinary method.

According to the present invention, soyasapogenol B can be obtained byallowing a culture containing a protein having saponin-decomposingactivity, a protein according to the present invention or a proteinobtained from a host according to the present invention to act on asubstance containing a glycoside having soyasapogenol B as an aglyconeand/or said glycoside.

More specifically, for example, about 1% to 10% by weight saponin(Koshiro Seiyaku) is dissolved in water or a buffer solution, such as anacetate buffer or a phosphate buffer, to which saponin-decomposingenzyme is added. The reaction is carried out at an appropriatetemperature, for example 20° C. to 50° C., after which the resultingreaction solution is extracted with an organic solvent such as ethylacetate to obtain soyasapogenol B.

EXAMPLES

The present invention is further illustrated by the following examplesthat are not intended as a limitation of the invention.

Reference Example 1 Confirmation of Aspergillus Saponin-DecomposingEnzyme

A PDA slant (about 1 cm²) of Aspergillus sp. PF1224 (PERM BP-8004) wasinoculated into 100 ml of a TS medium (2.0% soluble starch, 1.0%glucose, 0.5% polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2%soybean grounds, and 0.2% calcium carbonate (pH 7.0 beforesterilization)) dispensed into a 500-ml Erlenmeyer flask. Incubation wasthen carried out at 25° C. for 3 days with shaking. The resultingculture (4 ml) was inoculated into 100 ml of an MY medium (4% maltextract, 2.0% yeast extract, 0.2% potassium dihydrogenphosphate, 0.2%ammonium sulfate, 0.03% magnesium sulfate heptahydrate, 0.03% calciumchloride dihydrate (pH 7.0)) supplemented with 4.0% soybean saponin(Koshiro Seiyaku) dispensed into a 500-ml Erlenmeyer flask andincubation was then carried out for 3 days with shaking.

Saponin-decomposing activity shown in Test Example 1 was used as anindex in the purification of saponin-decomposing enzyme derived fromAspergillus hereinafter.

The resulting culture (about 800 ml) was filtered with a glass filter(G3) and then centrifuged (8,000 rpm, 30 minutes) to remove cell debris.Ammonium sulfate (294 g) was added to about 570 ml of the supernatantthus obtained and the resulting precipitate was recovered bycentrifugation (8,000 rpm, 30 minutes). This precipitate was dissolvedin about 120 ml of a buffer solution A (0.1 M sodium acetate buffer, 1 Mammonium sulfate (pH 5.8)) and the resulting solution was subjected tohydrophobic chromatography using Butyl Toyopearl 650S (26 mm i.d.×330mm) (Tosoh Co.). Elution was carried out with a concentration gradientfrom a buffer solution B (0.1 M sodium phosphate buffer-1 M ammoniumsulfate (pH 5.8)) to 0.1 M sodium phosphate buffer (pH 5.8) and anunadsorbed fraction and a fraction eluted with an ammonium sulfate at aconcentration from 1 M to 0.5 M were recovered.

Each of the recovered fractions was concentrated using Pellicon XL(cut-off molecular weight: 10,000) (Millipore), after which 1 M Tris-HClbuffer and ammonium sulfate were added to the concentrate so as to maketheir concentration the same as in a buffer solution C (50 mM Tris-HClbuffer, 1 M ammonium sulfate (pH 7.5)) and the resulting solution wassubjected to hydrophobic chromatography using 6 ml of Resource PHE(Amersham Biosciences). Elution was carried out with a concentrationgradient from the buffer solution C to the 50 mM Tris-HCl buffersolution (pH 7.5) and an unadsorbed fraction was recovered.

The fraction thus obtained was concentrated using Ultrafree 15 (cut-offmolecular weight: 5,000) (Millipore) and then subjected to gelfiltration chromatography using Superdex 200 pg (16 mm i.d.×600 mm)(Amersham Biosciences). Elution was carried out with a buffer solution D(25 mM sodium phosphate buffer, 0.15 M sodium chloride (pH 5.8)) and afraction of a cut-off molecular weight of about 90 kDa was recovered.

SDS-PAGE was carried out with this fraction and a single band with anestimated molecular weight of about 90 kDa was observed.

Test Example 1 Measurement of Saponin-Decomposing Activity

An enzyme solution containing an enzyme of interest was desalted using aPD-10 column (Amersham Biosciences), after which an equal volume of 2%saponin solution was mixed and reaction was carried out at 37° C. forabout 16 hours. The resulting reaction solution was extracted with anequal volume of ethyl acetate and the resulting extract was developedusing TLC (solvent system used: chloroform:methanol=95:5). Utilizingcolor reaction of vanillin-sulfuric acid, soyasapogenol B having an Rfvalue of 0.35 was detected to measure enzyme activity of the enzymesolution of interest.

Test Example 2 Quantitative Analysis of Saponin-Decomposing Activity

A diluted enzyme solution was added to 50 μl of a 2% saponin solution tomake a total volume of 100 μl and the resulting admixture was reactedfor 30 minutes. Next, the resulting reaction solution was extracted withan equal volume of ethyl acetate and a 50 μl portion of the extract wasdiluted with 450 μof mobile phase. A 10 μl portion of the dilution wassubjected to high performance liquid chromatography under the followingconditions and a peak height at a retention time of about 7.5 minuteswas measured. By comparing this height with that of authenticsoyasapogenol B, saponin-decomposing activity of this enzyme wasquantitatively evaluated.

-   -   Column: Inertsil ODS −2, 5 μm (4.6 mm i.d×250 mm)    -   Column temperature: 40° C.    -   Mobile phase: acetonitrile:methanol:water=50:35:15    -   Mobile phase flow rate: 0.8 ml/min

Example 1 Isolation and Purification of Saponin-Decomposing EnzymeDerived from genus Neocosmospora (SDN)

A PDA slant (about 1 cm²) of Neocosmospora vasinfecta var. vasinfectaPF1225 (FERM BP-7475) was inoculated into 100 ml of a TS mediumdispensed into a 500-ml Erlenmeyer flask. Incubation was carried out at25° C. for 3 days with shaking. The resulting culture (4 ml) wasinoculated into 100 ml of an MY medium supplemented with 4.0% soybeansaponin (Koshiro Seiyaku) dispensed into a 500-ml Erlenmeyer flask andthen incubation was carried out for 3 days with shaking.

Saponin-decomposing activity shown in Test Example 1 was used as anindex in the purification of saponin-decomposing enzyme derived fromgenus Neocosmospora hereinafter.

The resulting culture (about 800 ml) was diluted with about 2 timesvolume of water and then centrifuged (8,000 rpm, 30 minutes) to removecells. Ammonium sulfate (171 g) was added to the supernatant and theresulting precipitate was removed by centrifugation (8,000 rpm, 30minutes). Further ammonium sulfate (573 g) was added to the resultingsupernatant and the resulting precipitate was recovered bycentrifugation (8,000 rpm, 30 minutes) and dissolved in 70 ml of abuffer solution C. The resulting solution was subjected to hydrophobicchromatography using Butyl Toyopearl 650S (26 mm i.d.×110 mm) (TosohCo.). Elution was carried out with a concentration gradient from abuffer solution C to 50 mM Tris-HCl buffer (pH 7.5) and an unadsorbedfraction was recovered.

Ammonium sulfate (about 239 g) was added to about 500 ml of thisfraction and the resulting precipitate was recovered by centrifugation.The recovered precipitate was then dissolved in 4 ml of a buffersolution B and the resulting solution was subjected to hydrophobicchromatography using Phenyl Sepharose FF (16 mm i.d.×100 mm) (AmershamBiosciences). Elution was carried out with a concentration gradient froma buffer solution B to a 0.1 M sodium phosphate buffer solution (pH 5.8)and a fraction at an ammonium sulfate concentration of about 0.4 M wasrecovered.

The fraction thus recovered was subjected to gel filtrationchromatography using Superdex 200 pg (16 mm i.d.×600 mm) (AmershamBiosciences). Elution was carried out with a buffer solution E (50 mMTris-HCl buffer, 0.15 M sodium chloride (pH 7.5)) and a fraction of acut-off molecular weight of about 76,000 was recovered.

SDS-PAGE was carried out with this fraction and a single band of anestimated molecular weight of about 77 kDa was observed.

Example 2 Amino Acid Sequence Analysis of Saponin-Decomposing Enzyme(SDN)

2a) Amino Acid Sequence of the N-Terminal Side

The fraction prepared as in Example 1 was subjected to SDS-PAGE andblotted onto a PVDF membrane (Immobilon-PSQ) (Millipore), after whichthe membrane was washed and dried in air. This was subjected to aprotein sequencer model 492 (Applied Biosystems) to analyze the aminoacid sequence.

The amino acid sequence obtained by the analysis was as follows:

-   N-terminal amino acid sequence: ASPPASVPNNPSSEEITLQ (SEQ ID NO: 7)

2b) Analysis of Inner Amino Acid Sequence (Peptide Mapping)

The fraction prepared in Example 1 was subjected to SDS-PAGE and theresultant proteins were stained using Coomassie Brilliant Blue R250. Asingle band stained at an estimated molecular weight of about 77 kDa wasexcised and destained using a 0.2 M ammonium bicarbonate buffer solution(pH 8.0) in 50% acetonitrile and dried at room temperature for about 2hours in air.

Next, this gel strip was immersed in a 0.2 M ammonium bicarbonate buffersolution (pH 8.0) containing 0.02% Tween 20, after which trypsin(Promega) was added and reaction was carried out at 37° C. for 2 days.After the reaction, the supernatant was recovered and the gel strip wasfurther washed 3 times with 60% acetonitrile and 0.1% trifluoraceticacid. The resulting washings and the reaction supernatant were combined,concentrated and subjected to a Model 172μ preparative HPLC system(Applied Biosystems) (RP-300 Aquiapore C18, 220×2.1 mm, with aconcentration gradient from 0.1% trifluoracetic acid-35% acetonitrile to0.085% trifluoracetic acid-35% acetonitrile). The following 5polypeptides were fractionated.

Trp26.8: LVFNPSPK (SEQ ID NO: 8) Trp27.59: WNVAADGSGPSGEIR (SEQ ID NO:9) Trp32.07: VTILHNPEGVAPITAK (SEQ ID NO: 10) Trp39.43:EHSDTIPWGVPYVPGSQ (SEQ ID NO: 11) Trp41.3: LTDYSFDWYSDIR (SEQ ID NO: 12)

Example 3 Cloning and Sequence Analysis of Saponin-Decomposing Enzyme(SDN)

3a) Preparation of Long Probe Using PCR

A genomic DNA was prepared from cultured cells of Neocosmosporavasinfecta var. vasinfecta PF1225 (FERM BP-7475) to be used as atemplate for PCR.

The genomic DNA was isolated according to the method of Horiuchi et al.(J. Bacteriol., 170, 272-278, 1988). First, cells cultured in a TSmedium were recovered by centrifugation (7,500 rpm, 10 minutes). Thecells thus obtained were lyophilized, suspended in a TE solution (10 mMTris-HCl buffer, 1 mM EDTA (pH 8.0)) and then treated in a 3% SDSsolution at 60° C. for 30 minutes. Then TE-saturated phenol extractionwas carried out to remove cell debris.

The extract was precipitated with ethanol and treated with RibonucleaseA (Nippon Gene) and Proteinase K (Wako Pure Chemical Industries, Ltd.),and the nucleic acid was then precipitated with 12% polyethylene glycol6000. The precipitate was subjected to TE-saturated phenol extractionand ethanol precipitation, and the resulting precipitate was dissolvedin a TE solution to obtain the genomic DNA.

Based on the peptide sequences obtained in Example 2, the followingoligonucleotides encoding these sequences were synthesized and used asprimers for PCR:

Primer N1: CCIGCITCNGTNCCNAA (SEQ ID NO: 13) Primer N2:CCIGCIAGYGTNCCNAA (SEQ ID NO: 14) Primer 2A: CCRTCIGCNGCNACRTT (SEQ IDNO: 15) Primer 3A: CCCCAIGGDATNGTRTC (SEQ ID NO: 16) Primer 4A:ACICCYTCNGGRTTRTG (SEQ ID NO: 17)

The PCR was carried out using Takara Taq (Takara Shuzo Co., Ltd.). Thefragments were amplified by repeating 10 cycles of 30 seconds at 94° C.,30 seconds at 45° C., and 3 minutes at 55° C., after heat denaturationat 94° C. for 1 minute, followed by 20 cycles of 30 seconds at 94° C.,30 seconds at 47° C., and 3 minutes at 60° C. As a result, a specificfragment of about 0.8 kb was amplified in a combination of primer N1 andprimer 4A. This fragment was cloned into pCR2.1-TOPO (pCR2.1-2) using aTOPO TA cloning kit (Invitrogen).

DNA sequence analysis was carried out using dRhodamine Terminator cyclesequencing ready reaction (Applied Biosystems) and ABI PRISM 310 geneticanalyzer (Applied Biosystems). Decoding of the PCR product cloned inpCR2.1-2 revealed that this fragment was the amplification of the regionfrom position 88 to position 812 of the sequence of SEQ ID NO: 1.

3b) Southern Analysis and Sequence Decoding Using Inverse PCR

In Southern analysis, a genomic DNA digested with EcoRI was subjected toagarose gel electrophoresis and then to blotting onto Hibond N+(Amersham Biosciences). An ECF Random-Prime Labelling and DetectionSystem (Amersham Biosciences) was used for hybridization and a MolecularImager FX (Bio-Rad) was used for band detection.

A band of about 2 kb was detected when the PCR product obtained in 3a)in Example 3 was used as a probe.

Next, the genomic DNA was digested with EcoRI, and a fragment of about 2kb was recovered and circularized using a DNA ligation kit ver. 2(Takara Shuzo Co., Ltd.). Using the resulting loop as a template, thefragment was amplified using LA Taq (Takara Shuzo Co., Ltd.) with thefollowing primers for inverse PCR by repeating 25 cycles of a serialstep consisting of 30 seconds at 94° C., 30 seconds at 50° C., and 4.5minutes at 72° C., after heat denaturation at 94° C. for 1 minute. Thisamplified fragment of about 2 kb was cloned using a TOPO TA cloning kit(Invitrogen) and its sequence was analyzed using primer walking.

Primer 1 for inverse PCR: TGACGCTGATACCAACCGCG (SEQ ID NO: 18) Primer 2for inverse PCR: CTAGTGGCAGTATTGGACAG (SEQ ID NO: 19)

3c) Determination of Translation Region Using 3′ RACE and 5′ RACEMethods

The translation region was determined by the 3′ RACE and 5′ RACE methodsusing cDNA as a template. cDNA was prepared as follows.

As described in Example 1, a one ml portion of the culture ofNeocosmospora vasinfecta var. vasinfecta PF1225 (FERM BP-7475) culturedin a TS medium was inoculated into 100 ml of an MY medium supplementedwith 1% soybean saponin dispensed into a 500-ml Erlenmeyer flask. Afterincubation at 25° C. for 32 hours with shaking, cells were filteredthrough a nylon mesh (50 μm) and the cells thus obtained were frozenwith liquid nitrogen. The frozen cells were smashed with a mortar andpestle and the whole RNA was extracted from the smashed cells. The wholeRNA was extracted using ISOGEN (Nippon Gene) according to the attachedprotocol. mRNA was purified from the whole RNA using Oligotex-dT30<Super> (Roche Diagnostics) according to the attached protocol.

By applying a 5′/3′ RACE kit (Roche Diagnostics) to this mRNA, 3′ and 5′regions were amplified using 3′ RACE and 5′ RACE according to theattached protocol. In this case, in the 3′ RACE method, AmpriTaq Gold(Applied Biosystems) was used in the primary PCR and PCR Supermix HighFidelity (Lifetech Oriental Co., Ltd.) was used in the secondary PCR. Inthe primary and secondary PCRs in the 5′ RACE method, PCR Supermix HighFidelity (Lifetech Oriental Co., Ltd.) was used.

The sequences of 3′ RACE- and 5′ RACE-specific primers were as follows.

3′ RACE specific primer for primary PCR: CCCAGGCCTTTAAGGATGGC (SEQ IDNO: 20) 3′ RACE specific primer for secondary PCR: CTAGTGGCAGTATTGGACAG(SEQ ID NO: 19) 5′ RACE specific primer for cDNA synthesis:TGACGCTGATACCAACGGCG (SEQ ID NO: 18) 5′ RACE specific primer for primaryPCR: CTGCTTGAGGGTAATGGGCTC (SEQ ID NO: 21) 5′ RACE specific primer forsecondary PCR: ACAGACGCCGGAGGAGAAGCG (SEQ ID NO: 22)

The translation region of the SDN gene shown in SEQ ID NO: 1 was thusdetermined. From the result in Example 2a), the N terminal of the matureprotein of SDN amino acid sequence was found to be located at position27 from Met of the translation initiation site.

The presence of introns in the translation region was confirmed bycomparing DNA sequences of the genomic DNA and cDNA. It was revealedthat no intron was present in this translation region.

Example 4 Expression of Saponin-Decomposing Enzyme (SDN) in Escherichiacoli

PCR was carried out with primers for expression in E. coli shown belowusing the cDNA obtained in Example 3 as a template.

The translation region of the SDN gene was amplified using PCR SupermixHigh Fidelity (Lifetech Oriental Co., Ltd.) by repeating 25 cycles of aserial step consisting of 30 seconds at 94 C, 30 seconds at 50 C, and 2minutes at 72 C, after denaturation at 94° C. for 1 minute. A fragmentobtained by digesting the resulting product with restriction enzymesNdeI and BamHI was ligated to plasmid pET15b (Novagen, Inc.) digestedwith the same restriction enzymes, using a DNA ligation kit ver. 2(Takara Shuzo Co., Ltd.). E. coli strain BL21 (DE3) was transformedusing this product according to an ordinary method and colonies havingampicillin resistance were obtained.

N-terminal primer for E. coli expression: GGGCATATGGCTTCTCCTCCTGCTTCTG(SEQ ID NO: 23) C-terminal primer for E. coli expression:GGGGGATCCTTAAGTGCCGCTCTGAGGACTACG (SEQ ID NO: 24)

Colonies obtained were used for the following experiment.

Cells taken from the colonies were inoculated into 50 ml of an LB mediumcontaining 50 μg/ml ampicillin dispensed in a 250-ml Erlenmeyer flaskand incubation was carried out at 37° C. overnight with shaking. A 2 mlportion of this culture was further inoculated into 50 ml of an LBmedium containing 50 μg/ml ampicillin dispensed in a 250-ml Erlenmeyerflask and incubation was carried out at 37° C. for 3 hours with shaking.Isopropyl-β-D-thiogalactopyranoside was added to the resulting cultureat a final concentration of 0.4 mM and incubation was carried out for 3hours for induction.

Cells thus cultured were collected by centrifugation and suspended in abuffer solution F (50 mM Tris-HCl buffer, 2 mM EDTA (pH 8.0)), afterwhich cells were again recovered by centrifugation. After freezing at−80° C., these cells were suspended in 5 ml of a buffer solution F,lysozyme and Triton X 100 were added at final concentrations of 100μg/ml and 0.1%, respectively, and the resulting suspension was allowedto stand at room temperature for about 20 minutes. Under ice cold, thecells were disrupted by ultrasonic treatment twice at a 50% duty cyclefor 30 seconds using Sonifier 450 (BRANSON) and cell debris were removedby centrifugation.

Saponin-decomposing activity was measured according to Test Example 1for the cell extract thus obtained as a crude enzyme solution.

As a result, a spot of the decomposed product, soyasapogenol B, wasobserved on TLC only for the extract of the cells in which thetranslation region of the saponin-decomposing enzyme was cloned.

Example 5 Expression of Saponin-Decomposing Enzyme (SDN) in Trichodermaviride

5a) Construction of Vector for Transformation

PCR was carried out as described in Example 4 with primers forexpression in Trichoderma shown below using the cDNA obtained in Example3 as a template.

A fragment obtained by digesting the resulting PCR product withrestriction enzymes SmaI and PstI was ligated to plasmid pCB1-M2 (seeExample 5 of WO 98/11239) digested with StuI and PstI, using a DNAligation kit ver. 2 (Takara Shuzo Co., Ltd). The product was digestedwith XbaI, dephosphorylated and then linked to an XbaI cassette of thepyr4 gene derived from Neurospora crassa to construct pCB-SBe (FIG. 1).

N-terminal primer for expression in Trichoderma: (SEQ ID NO: 25)GGGCCCGGGGCGCATCATGCACTTCTTTGACAAAGCGAC C-terminal primer for expressionin Trichoderma: (SEQ ID NO: 26) GGGCTGCAGTTAAGTGCCGCTCTGAGGACT

The XbaI cassette of the pyr4 gene was constructed as follows.

pFB6 (Biochem. Biophys. Res. Commun., 112, 284-289, 1983) was firstdigested with BGlII and then partially digested with HindIII to recovera fragment of about 1.9 kb. This fragment was ligated to pLITMUS28 (NewEngland Biolabs) digested with BglII and HindIII. Next, the product wasdigested with BglII, blunted using a DNA blunting kit (Takara Shuzo Co.,Ltd.), and then linked to a phosphorylated linker pXbaI (Takara ShuzoCo., Ltd.) to construct the XbaI cassette of the pyr4 gene.

5b) Acquisition of Uracyl-Requiring Strain Derived from Trichodermaviride

A spore suspension of Trichoderma viride MC300-1 (about 1.0×10⁹ CFU/ml)was exposed to 2 UV lights at a distance of 30 cm with gentle mixing.The suspension was spread on a selective medium and incubated at 28° C.for 7 days and then grown strains were selected.

A selective medium used was a minimum medium (0.2% potassiumdihydrogenphosphate, 0.4% ammonium sulfate, 0.03% urea, 0.03% magnesiumsulfate heptahydrate, 0.03% calcium chloride, 0.5% glucose, 2.5% agar,0.01% trace elements (5 mg of ion sulfate heptahydrate, 1.56 mg ofmanganese sulfate heptahydrate, 1.4 mg of zinc sulfate heptahydrate, and2.0 mg of cobalt chloride dissolved in 1 L of water) supplemented with10 μg/ml uridine and 1.0 mg/ml 5-fluoroorotic acid.

5c) Transformation of Trichoderma viride and Detection ofSaponin-Decomposing Activity of Each Recombinant

Cells of the uracyl-requiring Trichoderma viride strain obtained in 5b)of Example 5 were inoculated into 50 ml of a cell forming medium (1.0%yeast extract, 1.0% molt extract, 2.0% polypeptone, 2.5% glucose, 0.1%potassium dihydrogenphosphate, 0.05% magnesium sulfate heptahydrate, (pH7.0 before sterilization)) dispensed in a 200-ml Erlenmeyer flask andincubation was carried out at 28° C. for 2 days with shaking. Myceliawere recovered from the resultant culture by centrifugation. Next,protoplasts were prepared from the mycelia, after which a DNA solutionof plasmid pCB-SBe was added to carry out transformation (see Example 7of WO 98/11239).

Further, in regeneration of transformants, 0.5 M sucrose was added tothe minimum medium. Grown colonies were again inoculated onto theminimum medium and colonies grown in the medium were recognized astransformants.

Plasmid pCB-SBe was introduced into the uracyl-requiring Trichodermaviride strain. As a result, 3 transformants per 1 μg of pCB-SBe wereobtained. Each of the transformants, 25 strains, was cultured (seeExample 1 of WO 98/11239) and the resultant culture supernatant wassubjected to SDS-PAGE, on which a band showing a molecular weight ofabout 68 kDa was observed only for the transformants.

Saponin-decomposing activity was measured according to Test Example 1for this culture supernatant. As a result, a spot of the decomposedproduct, soyasapogenol B, was observed on TLC. On the other hand, thisspot was not observed for the parent strain, the uracyl-requiringTrichoderma viride.

5d) Purification of Recombinant Saponin-Decomposing Enzyme (RecombinantSDN), and Comparison of Its Activity with Wild-Type Saponin-DecomposingEnzyme Derived from Neocosmospora vasinfecta var. vasinfecta PF1225(Wild-Type SDN)

The culture obtained in 5c) of Example 5 (about 700 ml) was centrifuged(8,000 rpm, 30 minutes) to remove cell debris. Ammonium sulfate (64 g)was added to about 560 ml of the supernatant thus obtained and theresulting precipitate was removed by centrifugation (8,000 rpm, 30minutes). Further, 74 g of ammonium sulfate were added to about 600 mlof the resultant supernatant and the resultant precipitate wasrecovered. To this precipitate were added 100 ml of 0.05 M Tris-HClbuffer (pH 7.5) and 16 g of ammonium sulfate and the admixture wassubjected to hydrophobic chromatography using Butyl Toyopearl 650S (26mm i.d.×330 mm) (Tosoh Co.). Elution was carried out with aconcentration gradient from a buffer solution C to 50 mM Tris-HCl buffer(pH 7.5) and an unadsorbed fraction and a fraction eluted at an ammoniumsulfate concentration from 1 M to 0.6 M were recovered.

Each of the fraction thus obtained was concentrated using Pellicon XL(cut-off molecular weight: 10,000) (Millipore). To about 8 ml of thisconcentrate were added 1.3 g of ammonium sulfate and 2 ml of 0.5 Msodium phosphate buffer (pH 5.8) and the admixture was subjected tohydrophobic chromatography using 6 ml of Resource PHE (AmershamBiosciences). Elution was carried out with a concentration gradient froma buffer solution B to 0.1 M sodium phosphate buffer (pH 5.8) and anunadsorbed fraction was recovered.

The fraction thus obtained was concentrated using Ultrafree 15 (cut-offmolecular weight: 5,000) (Millipore). This concentrate was subjected togel filtration chromatography using Superdex 200 pg (16 mm i.d.×600 mm)(Amersham Biosciences). Elution was carried out with a buffer solution G(50 mM sodium phosphate buffer, 0.15 M sodium chloride (pH 7.0)) and afraction of a cut-off molecular weight of about 68,000 was recovered.

SDS-PAGE was carried out with this fraction and a single band with anestimated molecular weight of about 68 kDa was observed.

The optimum pH and the optimum temperature for the recombinantsaponin-decomposing enzyme thus purified (occasionally referred to as“recombinant SDN” hereinafter) and the saponin-decomposing enzymepurified in Example 1 (occasionally referred to as “wild-type SDN”hereinafter) were measured according to Test Example 2.

In measuring the optimum pH, first, to 50 μl of a 2% saponin solutionwere added 20 μl each of 0.5 M individual buffer solutions (sodiumacetate buffer (pH 4.5, pH 5.0, pH 5.8); sodium phosphate buffer (pH5.0, pH 5.8, pH 7.0) and Tris-HCl buffer (pH 7.0, pH 8.0, pH 9.0)) and adiluted enzyme solution to make a total volume of 100 μl. Reaction wascarried out at 37° C. for 30 minutes, and then the amount ofsoyasapogenol B produced was measured.

Results are shown in FIG. 2.

In measuring the optimum temperature, first, to 50 μl of a 2% saponinsolution were added 20 μl of 0.5 M sodium phosphate buffer (pH 5.8) anda diluted enzyme solution to make a total volume of 100 μl. Reaction wascarried out at each specified temperature for 30 minutes, and then theamount of soyasapogenol B produced was measured.

Results are shown in FIG. 3.

As evident from these results, there was not much difference in activityalthough there was some difference in the molecular weight determined bySDS-PAGE.

Example 6 Amino Acid Sequence Analysis for Saponin-Decomposing EnzymeDerived from Aspergillus sp. PF1224 (SDA)

Saponin-decomposing enzyme purified from Aspergillus sp. PF1224 (FERMBP-8004) (SDA) (Reference Example 1) was fragmented as described in 2b)of Example 2 after excising a band of about 90 kDa, and subjected toHPLC as described in 2b) in Example 2 to fractionate the following 4kinds of peptides.

Trp23.67: LYNPDSPQPISAK (SEQ ID NO: 27) Trp24.0s: LQFNPAPK (SEQ ID NO:28) Trp38.05: VDWFSDLTSTGQVTGSK (SEQ ID NO: 29) Trp24.5: GEVSGSASVSIIHD(SEQ ID NO: 30)

Example 7 Cloning and Sequence Analysis of SDA Gene

7a) Preparation of Long Probe Using PCR

A genomic DNA was isolated from cells cultured as described in ReferenceExample 1, as described in Example 3.

Based on the peptide sequences obtained in Example 6, the followingoligonucleotides encoding these sequences were synthesized and used asprimers for PCR.

Primer 23.67s1: TAYAAYCCIGAYTCNCC (SEQ ID NO: 31) Primer 23.67s2:TAYAAYCCNGAYAGYCC (SEQ ID NO: 32) Primer 24.0s: CARTTYAAYCCIGCNCC (SEQID NO: 33) Primer 24.0a: GGIGCNGGRTTRAAYTG (SEQ ID NO: 34) 3 PrimerAARTCNGARAACCARTC (SEQ ID NO: 35) 38.05a1: Primer AARTCRCTRAACCARTC (SEQID NO: 36) 38.05a2:

The PCR was carried out as described in 3a) in Example 3.

As a result, a fragment of about 1 kb was specifically amplified in acombination of primer 24.0 s and primer 38.05a1 among the primers above,which was then cloned into pCR2.1-TOPO using a TOPO TA cloning kit(Invitrogen) (pSDAPCR1).

Results of sequence analysis revealed that the fragment cloned intopSDAPCR1 was the amplification of the region from position 709 toposition 1748 of the sequence of SEQ ID NO: 3.

7b) Southern Analysis and Screening using E. coli Colony Library

In Southern analysis, a genomic DNA previously digested with BamHI,EcoRI, and HindIII was subjected to agarose gel electrophoresis and thento blotting onto Hibond N+ (Amersham Bioscience). An ECF Random-PrimeLabelling and Detection System (Amersham Bioscience) was used forhybridization and a Molecular Imager FX (Bio-Rad) was used for banddetection.

Bands for a BamHI fragment of about 10 kb, an EcoRI fragment of about 20kb, and a HindIII fragment of about 5 kb were detected when the PCRproducts obtained in 7a) above were used as a probe.

Next, the genomic DNA was digested with HindIII, and fragments of about4 kb to 6 kb were recovered. The product was then linked to pUC18, whichwas previously digested with HindIII and dephosphorylated, to transforman E. coli strain DH5a. This E. coli was grown on an LB agar mediumsupplemented with ampicillin for colony formation, about 1,000 coloniesthus obtained were blotted onto Hibond N+ (Amersham Bioscience). Here,one kind of positive clone (pSDAHind5/18) was obtained using the PCRproduct obtained in the abovementioned 7a) as a probe. This clonecontained a HindIII fragment of about 5 kb.

7c) Determination of Translation Region Using 3′ RACE and 5′ RACEMethods

As described in 3c) in Example 3, the whole RNA was extracted fromculture cells of Aspergillus sp. PF1224 (FERM BP-8004) prepared inReference Example 1 and further, mRNA was isolated using a QuickPrepmRNA purification kit (Amersham Bioscience) according to the attachedprotocol.

By applying a 5′/3′ RACE kit (Roche Diagnostics) to this mRNA, 3′ and 5′regions were amplified. LA Taq (Takara Shuzo Co., Ltd.) was used foreach PCR.

Sequences of 3′ RACE- and 5′ RACE-specific primers were as follows.

3′ RACE specific primer for primary PCR: CCTCGATACCCGAGGGACCG (SEQ IDNO: 37) 3′ RACE specific primer for secondary PCR: GATGGGTTGCATGTTATCGC(SEQ ID NO: 38) 5′ RACE specific primer for cDNA synthesis:GCGATAACATGCAACCCATC (SEQ ID NO: 39) 5′ RACE specific primer for primaryPCR: GACCACCTGGTTCAGTGGTG (SEQ ID NO: 40) 5′ RACE specific primer forsecondary PCR: GGGTTATAGAGTCTGGTAACG (SEQ ID NO: 41)

The translation region of the SDA gene shown in SEQ ID NO: 3 was thusdetermined. The SDA protein purified as Reference Example 1 was analyzedfor the mature N-terminal amino acid sequence in the same manner asdescribed in Example 2a). As a result, the N terminal of the matureprotein of SDA amino acid sequence was found to be located at position29 from Met of the translation initiation site. Further, the presence ofintrons in the translation region was confirmed by comparing DNAsequences of the genomic DNA and cDNA. It was revealed that no intronwas present in this translation region.

Example 8 Expression of Saponin-Decomposing Enzyme Derived fromAspergillus sp. PF1224 (SDA) in Trichoderma viride

8a) Construction of Vector for Transformation

First, PCR was carried out as described in Example 4 with primers forexpression in Trichoderma shown below using pSDAHind5/18 obtained inExample 7b as a template.

A fragment obtained by digesting the resulting PCR product withrestriction enzymes StuI and XhoI was ligated to plasmid pCB1-M2 (seeExample 5 of WO 98/11239) previously digested with StuI and XhoI using aDNA ligation kit ver. 2 (Takara Shuzo Co., Ltd.) to construct pCB-SDAe(FIG. 4)

N-terminal primer for SDA expression in Trichoderma: (SEQ ID NO: 42)GGGAGGCCTGCGCATCATGCATGTTGTCGCAAGTACCAC C-terminal primer for SDAexpression in Trichoderma: (SEQ ID NO: 43)GGGCTCGAGTACCTCAAGTCCCATTTGCCGGCTGC

8b) Transformation of Trichoderma viride and Detection ofSaponin-Decomposing Activity of Each Recombinant

A host, the uracyl-requiring Trichoderma viride strain obtained in 5b)in Example 5, was transformed by the co-transformation method usingpCB-SDAe and vector pPYR4 in which the pyr4 cassette was ligated topLITMUS28 (see 5a) in Example 5), as described in 5c) in Example 5. As aresult, about 12 strains of transformants per 1 μg of DNA were obtained.

Each of the transformants thus obtained was cultured (see Example 1 ofWO 98/11239) and the resultant culture supernatant was subjected toSDS-PAGE, on which a band showing a molecular weight of about 80 kDa wasobserved only for the transformants.

Saponin-decomposing activity was measured using this culture supernatantas described in Test Example 1. As a result, a spot of the decomposedproduct, soyasapogenol B, was observed on TLC.

8c) Purification of Recombinant Saponin-Decomposing Enzyme Derived fromAspergillus sp. PF1224 (Recombinant SDA), and Comparison of Its Activitywith Wild-Type Saponin-Decomposing Enzyme Derived from Aspergillus sp.PF1224 (Wild-Type SDA)

The culture obtained in 8b) above (about 600 ml) was centrifuged (8,000rpm, 30 minutes) to remove cell debris. Ammonium sulfate (57 g) wasadded to about 500 ml of the supernatant thus obtained and the resultingprecipitate was removed by centrifugation. Further, 64 g (40% saturationfraction) and then 70 g (60% saturation fraction) of ammonium sulfatewere added to this supernatant and the resultant precipitate wasdissolved in a 0.1 M sodium phosphate buffer solution (pH 5.8). To the60% saturation fraction was added ammonium sulfate to make a finalconcentration of 1M, and then the admixture was subjected to hydrophobicchromatography using Butyl Toyopearl 650S (26 mm i.d.×250 mm) (TosohCo.). Elution was carried out with a concentration gradient from abuffer solution A to a 0.1 M sodium phosphate buffer solution (pH 5.8)and a fraction eluted at an ammonium sulfate concentration from 0.9 M to0.2 M was recovered.

The fraction thus obtained was concentrated using Pellicon XL (cut-offmolecular weight: 10,000) (Millipore) and Ultrafree 15 (cut-offmolecular weight: 10,000) (Millipore), desalted using a PD-10 column(Amersham Bioscience) and then subjected to ion-exchange chromatographyusing 6 ml of Resource Q (Amersham Bioscience). Elution was carried outwith a concentration gradient from 50 mM Tris-HCl buffer (pH 7.5) to 50mM Tris-HCl buffer-0.5 M sodium chloride (pH 7.5), and an unadsorbedfraction and a fraction eluted at a salt concentration of 0.08 M wererecovered.

The fraction thus obtained was concentrated using Ultrafree 15 (cut-offmolecular weight: 10,000) (Millipore) and then subjected to gelfiltration chromatography using Superdex 200 pg (16 mm i.d.×600 mm)(Amersham Bioscience). Elution was carried out with a buffer solution Gand a fraction of a cut-off molecular weight of about 50 kDa wasrecovered.

SDS-PAGE was carried out with this fraction and a single band with anestimated molecular weight of about 80 kDa was observed. Further, thecut-off molecular weight on the gel filtration and the molecular weighton the SDS-PAGE were different probably because this protein wasadsorbed unspecifically to the carriers.

The optimum pH and the optimum temperature were measured for therecombinant SDA thus purified and the saponin-decomposing enzymepurified in Reference Example 1 (wild-type SDA), according to TestExample 2.

The optimum pH and the optimum temperature were measured as described in5d) in Example 5.

Results are shown in FIGS. 5 and 6.

As a result, it was revealed that although the recombinant SDA exhibitedlower specific activity in sodium phosphate buffer at pH 7 as comparedto the wild-type SDA, it exhibited improved specific activity inTris-HCl buffer and at high pHs.

Example 9 Isolation and Purification of Saponin-Decomposing EnzymeDerived from Eupenicillium brefeldianum PF1226 (SDE)

A PDA slant (about 1 cm²) of Eupenicillium brefeldianum PF1226 (FERMBP-7476) was inoculated into 100 ml of a TS medium dispensed into a500-ml Erlenmeyer flask and then incubation was carried out at 25° C.for 3 days with shaking. The resulting culture (4 ml) was inoculatedinto 100 ml of an MY medium supplemented with 1.0% soybean saponin(Koshiro Seiyaku) dispensed into a 500-ml Erlenmeyer flask and thenincubation was carried out for 7 days with shaking.

The resulting culture (about 1,000 ml) was filtered with a glass filter(G3) to remove cell debris. Ammonium sulfate (73 g) was added to thisculture supernatant (about 640 ml) and the resulting precipitate wasremoved by centrifugation (8,000 rpm, 30 minutes). Further, ammoniumsulfate (256 g) was added to the resulting supernatant (about 670 ml)and the resulting precipitate was recovered by centrifugation. Thisprecipitate was dissolved in about 50 ml of 0.1 M sodium acetate buffer(pH 5.8), 13.2 g of ammonium sulfate was added, and then water was addedto make a final concentration of 1 M ammonium sulfate-0.1M sodiumacetate buffer.

After centrifugation, this solution was subjected to hydrophobicchromatography using Butyl Toyopearl 650S (26 mm i.d.×330 mm) (TosohCo.). Elution was carried out with a concentration gradient from abuffer solution C to 50 mM Tris-HCl buffer and a fraction eluted at anammonium sulfate concentration of 0.7 M to 0.5 M was recovered.

The recovered fraction was concentrated using Pellicon XL (cut-offmolecular weight: 10,000) (Millipore), after which sodium phosphatebuffer and ammonium sulfate were added to make the concentrate havingthe same component as the buffer solution A and the resulting solutionwas subjected to hydrophobic chromatography using 6 ml of Resource PHE(Amersham Bioscience). Elution was carried out with a concentrationgradient from a buffer solution A to a 0.1 M sodium phosphate buffersolution (pH 5.8) and a fraction eluted from an ammonium sulfateconcentration of 1 M to 0.3 M was recovered.

This fraction was concentrated using Ultrafree 15 (cut-off molecularweight: 10,000) (Millipore), desalted using a PD-10 column (AmershamBioscience) and then subjected to ion-exchange chromatography using 6 mlof Resource Q (Amersham Bioscience). Elution was carried out with aconcentration gradient from 20 mM sodium phosphate buffer (pH 7.0) to 20mM sodium phosphate buffer-1 M sodium chloride (pH 7.0), and anunadsorbed fraction was recovered.

The fraction thus obtained was concentrated using Ultrafree 15 (cut-offmolecular weight: 10,000) (Millipore) and then subjected to gelfiltration chromatography using Superdex 200 pg (16 mm i.d.×600 mm)(Amersham Bioscience). Elution was carried out with a buffer solution Gand a fraction of a cut-off molecular weight of about 90 kDa wasrecovered.

SDS-PAGE was carried out with this fraction and a single band with anestimated molecular weight of about 90 kDa was observed.

Example 10 Analysis of Amino Acid Sequence of SDE

10a) Amino Acid Sequence of N-Terminal Side

The N-terminal side amino acid sequence of the fraction prepared inExample 9 was analyzed as described in 2a) in Example 2. As a result,the following amino acid sequence was obtained.

-   N-terminal amino acid sequence: STTPAPPQPEPI (SEQ ID NO: 44)

10b) Peptide Mapping

The SDE purified in Example 9 was fragmented after excising a band ofabout 90 kDa, as described in 2b) in Example 2. The fragments weresubjected to HPLC as described in 2b) in Example 2 and the following 3kinds of peptides were fractionated.

-   Trp20.73: ADPAFSPDGTR (SEQ ID NO: 45)-   Trp34.21: LHPDDTHMGWSSF (SEQ ID NO: 46)-   Trp36.26: GFSGAGDEILYIGSTR (SEQ ID NO: 47)

Example 11 Cloning and Sequence Analysis of SDE Gene

11a) Preparation of long probes using PCR

The genomic DNA was isolated from the cells cultured in Example 9, asdescribed in Example 3.

Based on the sequences of the peptides obtained in Example 10, thefollowing oligonucleotides encoding these sequences were synthesized andused as primers for PCR.

Primer Ns: CCICARCCNGARCCNAT (SEQ ID NO: 48) Primer 20.37a:CTRAAIGCNGGRTCNGC (SEQ ID NO: 49) Primer 34.21a: CCANCCCATRTGNGTRTC (SEQID NO: 50)

The PCR was carried out as described in 3a) in Example 3.

As a result, a fragment of about 1 kb was specifically amplified with acombination of primer Ns and primer 20.73a among the primers above. Thisfragment was cloned into pCR2.1-TOPO using a TOPO TA cloning kit(Invitrogen) (pSDEPCR5).

Results of sequence analysis revealed that the fragment cloned intopSDEPCR5was the amplification of the region from position 70 to position1247 of the sequence of SEQ ID NO: 5.

11b) Screening Using Southern Analysis and Phage Library

In Southern analysis, a genomic DNA digested with PstI, SphI and XhoIwas subjected to agarose gel electrophoresis and then to blotting ontoHibond N+ (Amersham Bioscience). An ECF Random-Prime Labelling andDetection System (Amersham Bioscience) was used for hybridization and aMolecular Imager FX (Bio-Rad) was used for band detection.

Bands of a PstI fragment of about 3 kb, an SphI fragment of about 4 kb,and an XhoI fragment of about 6 kb were detected when the PCR productobtained in 11a) above was used as a probe.

Next, the genomic DNA was partially digested with Sau3A1. The resultantproduct was then linked to λEMBL3/BamHI vector (Stratagene) and packagedusing a MaxPlax packaging extract kit (Epicentre Technologies). Theresultant phage library (about 5×10⁴ PFU) was blotted onto Hibond N+(Amersham Bioscience) and then 5 kinds of positive clones were obtainedusing the PCR fragment cloned in pSDEPCR5 as a probe, according to a DIGHi-Prime DNA Labelling and Detection Kit (Roche Diagnostics). Of theseclones, a XhoI fragment was recovered from a phage DNA containing the 6kb XhoI fragment and cloned into pBluescript. II KS+ (pSDEXho/IIKS+1).

The DNA sequence of the SDE translation region was determined as shownin SEQ ID NO: 5 by the transposon method using pSDEXho/IIKS+1 as atemplate.

As a result of Example 10a), the N terminal of the mature protein of SDEamino acid sequence was found to be located at position 18 from Met ofthe translation initiation site.

The presence of introns in the translation region was confirmed bycomparing the sequences of the genomic DNA and cDNA. It was revealedthat no intron was present in the translation region.

Example 12 Expression of Saponin-Decomposing Enzyme Derived fromEupenicillium brefeldianum PF1226 (SDE) in Trichoderma viride

12a) Construction of Vector for Transformation

PCR was carried out with primers for Trichoderma secretion shown belowusing pSDEXho/IIKS+1 obtained in Example 11 as a template, as describedin Example 4.

A fragment obtained by digesting the resultant PCR product withrestriction enzymes SmaI and XhoI was ligated to plasmid pCB1-M2 (seeExample 5 of WO 98/11239) previously digested with SmaI and XhoI, usinga DNA ligation kit ver. 2 (Takara Shuzo Co., Ltd) to construct pCB-SDEs(FIG. 7).

N-terminal primer for SDE secretion in Trichoderma: (SEQ ID NO: 51)GGGCCCGGGCTCAGACTACCCCGGCACCTCCTCAGCC C-terminal primer for SDEsecretion in Trichoderma: (SEQ ID NO: 52)GGGCTCGACTACCTCATGCACCATTGAGCGGCTGGTGG

12b) Transformation of Trichoderma viride and Detection ofSaponin-Decomposing Activity of Each Recombinant

Host cells of the uracyl-requiring Trichoderma viride strain obtained in5b) of Example 5 were transformed by the co-transformation method usingpCB-SDEs and vector pPYR4 in which the pyr4 cassette was linked topLITMUS28 (see 5a) above), as described in 5c) in Example 5. As aresult, about 28 strains of transformants per 1 μg of DNA were obtained.

Each of the transformants thus obtained was cultured (see Example 1 ofWO 98/11239) and the resultant culture supernatant was subjected toSDS-PAGE, on which a band with an estimated molecular weight of about 67kDa was observed only for the transformants.

Saponin-decomposing activity was measured as described in Test Example 1for this culture supernatant. As a result, a spot of the decomposedproduct, soyasapogenol B, was observed on TLC.

12c) Purification of Recombinant Saponin-Decomposing Enzyme Derived fromEupenicillium brefeldianum PF 1226 (Recombinant SDE), and Comparison ofIts Activity with the Wild-Type Saponin-Decomposing Enzyme Derived fromEupenicillium brefeldianum PF1226 (Wild-Type SDE)

The culture obtained in 12b) in Example 12 (about 900 ml) wascentrifuged (8,000 rpm, 30 minutes) to remove cell debris. Ammoniumsulfate (78.7 g) was added to about 690 ml of the supernatant thusobtained and the resulting precipitate was removed by centrifugation.Further, 88.6 g of ammonium sulfate (40% saturation fraction) were addedto the resultant supernatant and the resultant precipitate was dissolvedto make 120 ml of 1 M ammonium sulfate-0.1 M sodium phosphate buffer (pH5.8). A 20 ml portion of this solution was subjected to hydrophobicchromatography using Butyl Toyopearl 650S, (26 mm i.d.×330 mm) (TosohCo.). Elution was carried out with a concentration gradient from abuffer solution A to a 0.1 M sodium phosphate buffer solution and afraction eluted at an ammonium sulfate concentration from 0.2 M to 0 Mwas recovered.

The fraction thus obtained was concentrated using Pellicon XL (cut-offmolecular weight: 10,000) (Millipore) and Ultrafree 15 (cut-offmolecular weight: 5,000) (Millipore), desalted using a PD-10 column(Amersham Bioscience) and then subjected to ion-exchange chromatographyusing 6 ml of Resource Q (Amersham Bioscience) Elution was carried outwith a concentration gradient from 50 mM Tris-HCl buffer (pH 7.5) to 50mM Tris-HCl buffer-0.5 M sodium chloride (pH 7.5), and a fraction elutedat a salt concentration from 0 M to 0.1 M was recovered.

The fraction thus obtained was concentrated using Ultrafree 15 (cut-offmolecular weight: 5,000) (Millipore) and then subjected to gelfiltration chromatography using Superdex 200 pg (16 mm i.d.×600 mm)(Amersham Bioscience). Elution was carried out with a buffer solution Gand a fraction of a cut-off molecular weight of about 50 kDa wasrecovered.

SDS-PAGE was carried out with this fraction and a single band with anestimated molecular weight of about 67 kDa was observed. Further, thecut-off molecular weight on the gel filtration and the molecular weighton the SDS-PAGE were different probably because this protein wasadsorbed unspecifically to the carriers.

The optimum pH and the optimum temperature for the recombinant SDE thuspurified and the saponin-decomposing enzyme purified in Example 9(wild-type SDE) were measured according to Test Example 2.

The optimum pH and the optimum temperature were measured as described in5d) in Example 5.

Results are shown in FIGS. 8 and 9.

As a result, it was revealed that the recombinant SDE exhibited improvedactivity in a Tris-HCl buffer solution and also at high pHs as comparedto the wild-type SDE.

1. An isolated protein selected from the group consisting of: (a) aprotein comprising the amino acid sequence of SEQ ID NO: 2; (b) aprotein that has at least 50% homology to the protein comprising theamino acid sequence of SEQ ID NO: 2 and having saponin-decomposingactivity; and (c) a protein comprising a modified amino acid sequence ofthe amino acid sequence of SEQ ID NO: 2 that has one or more amino acidresidues deleted, substituted, inserted, or added and havingsaponin-decomposing activity. 2-20. (canceled)
 21. The isolated proteinaccording to claim 1, which is encoded by a polynucleotide selected fromthe group consisting of: (i) a polynucleotide consisting of a DNAsequence of SEQ ID NO: 1; (ii) a polynucleotide that has at least 70%homology to the polynucleotide consisting of the DNA sequence of (i) andencodes a protein having saponin-decomposing activity; (iii) apolynucleotide consisting of a modified DNA sequence of the sequence of(i) that has one or more bases deleted, substituted, inserted, or addedand encoding a protein having saponin-decomposing activity; and (iv) apolynucleotide that hybridizes with a DNA sequence of a polynucleotidecomprising the DNA sequence of (i) under stringent conditions andencodes a protein having saponin-decomposing activity.
 22. A method forproducing soyasapogenol B, which comprises decomposing a glycosidehaving soyasapogenol B as an aglycone using the protein of claim 1.